Physiological investigations of the influences of byproduct pathways on 3‐hydroxypropionic acid production in Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae can naturally synthesize 3‐hydroxypropionic acid (3‐HP), 1,3‐propanediol (1,3‐PD), and 2,3‐butanediol (2,3‐BD) from glycerol. However, biosynthesis of these industrially important chemicals is constrained by troublesome byproducts. To clarify the influences of byproducts on 3‐HP production, in this study, a total of eight byproduct‐producing enzyme genes including pmd, poxB, frdB, fumC, dhaT, ilvH, adhP, and pflB were individually deleted from the K. pneumoniae genome. The resultant eight mutants presented different levels of metabolites. In 24‐h shake‐flask cultivation, the adhP‐ and pflB‐deletion mutants produced 0.41 and 0.44 g/L 3‐HP, respectively. Notably, the adhP and pflB double deletion mutant K. pneumoniaeΔadhPΔpflB produced 1.58 g/L 3‐HP in 24‐h shake‐flask cultivation. When K. pneumoniaeΔadhPΔpflB was harnessed as a host strain to overexpress PuuC, a native aldehyde dehydrogenase (ALDH) catalyzing 3‐hydroxypropionaldehyde (3‐HPA) to 3‐HP, the resulting recombinant strain K. pneumoniaeΔadhPΔpflB(pTAC‐puuC) (pTAC‐puuC is PuuC expression vector) generated 66.91 g/L 3‐HP with a cumulative yield of 70.84% on glycerol in 60‐h bioreactor cultivation. Additionally, this strain showed 2.3‐, 5.1‐, and 0.67‐fold decrease in the concentrations of 1,3‐PD, 2,3‐BD, and acetic acid compared with the reference strain K. pneumoniae(pTAC‐puuC). These results indicated that the byproducts exerted differential impacts on the production of 3‐HP, 1,3‐PD, and 2,3‐BD. Although combinatorial elimination of byproduct pathways could reprogram glycerol flux, the enzyme 1,3‐propanediol oxidoreductase (DhaT) that catalyzes 3‐HPA to 1,3‐PD and the enzymes ALDHs, especially, PuuC are most pivotal for 3‐HP production. This study provides a deep understanding of how byproducts affect the production of 3‐HP, 1,3‐PD, and 2,3‐BD in K. pneumoniae.

Genome analysis to decipher syntrophy in the bacterial consortium ‘SCP’ for azo dye degradation

“…3-HPA is further fermented to 1,3-propanediol (PDO) by an NADH-associated 1,3-propanediol dehydrogenase (EC 1.1.1.202) [ 42 ]. The four enzymes involved in both pathways, i.e., glycerol dehydrogenase (EC 1.1.1.6), DHA kinase (EC 2.7.1.29), glycerol dehydratase (EC 4.2.1.30), and 1,3-propanediol dehydrogenase (EC 1.1.1.202) are encoded by dha regulon [ 43 ]. The expression of these enzymes is induced in the presence of DHA or glycerol [ 44 ].…”

“…An additional dehydrogenation pathway for glycerol utilization (i.e., glycerol degradation V) involves the conversion of glycerol to DHA by glycerol dehydrogenase, followed by phosphorylation of DHA to DHAP by a PEP-dependent DHA kinase (EC 2.7.1.121; encoded by dhaKL ) [ 42 , 43 ]. The genes for PEP-dependent DHA kinase (EC 2.7.1.121) were exclusively identified in the APG4 genome.…”

Significance and roles of synonymous codon usage in the evolutionary process of Proteus

“…Even though mutation pressure derived from nucleotide composition constraint is regarded as the dominant evolutionary dynamic in evolutionary trends of Proteus spp., other evolutionary dynamics, such as natural selection and fine‐tune translation, also modify codon usage‐derived evolutionary trends in these species. In previous studies, nucleotide usage variations are considered as an evolutionary dynamic for reflecting the effects of nucleotide composition constraint on synonymous codon usage patterns for microorganisms [27,30,42–46]. The four types of nucleotide base can serve as cornerstones for evolutionary trends of different organisms.…”

Rational Proteomic Analysis of a New Domesticated Klebsiella pneumoniae x546 Producing 1,3-Propanediol

“…In the whole TCA cycle, FumB and FrdB provide energy for bacteria. Therefore, the up-regulation of FumB and FrdB might accelerate cell growth and increase the yield of 1,3-PDO (Tseng et al, 2001;Xue et al, 2010;Huang et al, 2013;Li et al, 2019).…”

“…1,3-PDO can be produced by chemical synthesis or biosynthesis using Klebsiella pneumoniae. Because of its relatively high yield and low environmental pollution, K. pneumoniae is preferable to be used in 1,3-PDO production (Li et al, 2019;Mitrea and Vodnar, 2019;Zabed et al, 2019). Researchers have adopted several strategies such as the domestication method, genetic modification, medium optimization, and other methods to significantly increase the output of 1,3-PDO (Zhang et al, 2018;Lee et al, 2019;Ma et al, 2019;Zhou et al, 2019a).…”